PROJECT SUMMARY/ABSTRACT Immune tolerance normally functions to maintain mucosal homeostasis and when deficient is associated with inflammatory bowel disease (IBD). The current research proposal addresses the unanswered question of how carcinoembryonic antigen related cell adhesion molecule 1 (CEACAM1) and T-cell immunoglobulin mucin do- main 3 (TIM-3) act in specific parenchymal and hematopoietic cells in intestines to regulate immune tolerance and the pathogenesis and treatment of IBD. Our long-term goals are to define the biophysical nature of the in- teraction between CEACAM1 and TIM-3, delineate the relative role played by antigen presenting cells (APC) and T cells in determining CEACAM1 and TIM-3 mediated tolerance in intestinal inflammation, define the spe- cific elements of the regulatory milieu that CEACAM1 and TIM-3 control and especially that played by Epstein Barr virus induced gene 3 (EBI3)-related cytokines and guided by these insights draw nearer to the translation of these concepts by building on CEACAM1 Fc-fusion proteins as a platform for therapy. The objective of this research is to elucidate how, at the molecular and physiological levels, CEACAM1 and TIM-3 function in im- mune tolerance under homeostatic conditions and during pathologic responses associated with intestinal in- flammation. Our central hypothesis is that CEACAM1 and TIM-3 heterodimerize as ligands for each other such that TIM-3-mediated tolerance requires CEACAM1, and in the absence of CEACAM1, TIM-3 promotes inflam- mation. This rationale is derived from recent studies that CEACAM1 and TIM-3 form a biochemical association with each other and when CEACAM1 is deficient intestinal inflammation is unrestrained and anti-cancer im- munity is disabled even in the presence of excess TIM-3 expression. Our central hypothesis will be tested with four specific aims: 1) Determine the binding interface and mode of CEACAM1 and TIM-3 interactions. 2) De- fine the relative roles of CEACAM1 and TIM-3 on T cells and APCs in determining T cell inhibition and regula- tion. 3) Determine the functional role of CEACAM1 in the development of tolerance during intestinal inflamma- tion in relation to controlling regulatory versus inflammatory cytokines. 4) Determine the role of CEACAM1 or TIM-3 ligation by N-domain Fc fusion proteins in affecting T cell mediated colitis. In Aim 1, we will determine the molecular details of the CEACAM1-TIM-3 interaction. Aim 2 will define the specific roles of CEACAM1 and/or TIM-3 in T cells and APC in determining T cell responses to antigen and development of colitis. Aim 3 will characterize the identity of EBI3 expressing cells in intestinal mucosa and their relationship with CEACAM1 and TIM-3 expression on the APC and T cell. In Aim 4, we will examine the response of colitis to therapeutic intervention with Fc fusion proteins containing the functional ligand of CEACAM1 and TIM-3. Overall, this pro- posal is significant because not only will define the biophysical and molecular interactions between CEACAM1 and TIM-3 but will elucidate how this interaction functions in mucosal homeostasis and inflammation and whether it can therapeutically manipulated in a precise manner based upon the insights gained.